Identification of conserved hydrophobic C-terminal residues of the human papillomavirus type 1 E1E4 protein necessary for E4 oligomerisation in vivo.
Previous studies have shown that human papillomavirus (HPV) E4 proteins undergo oligomerisation, although the precise sequences involved have not been identified. Using the yeast two-hybrid system we have identified HPV 1 E4 sequences that are critical to multimerisation. Fusion proteins were created by linking wild-type and mutant E4 proteins to a LexA DNA-binding domain or a B42 transactivation domain. HPV 1 E4:E4 interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae. This assay showed that (1) amino acid residues 95 to 115 at the carboxy-terminus were critical for oligomerisation and (2) hydrophobic residues (isoleucine 107, phenylalanine 114) in this domain are major determinants in the formation of oligomers. Interestingly, the carboxy-terminal domain shares homology with other E4 proteins of cutaneous HPV types and, furthermore, positions 107 and 114 are conserved residues. Substitution of the conserved aspartate amino acids (residues 110 and 112) did not abrogate E4 oligomerisation. Chemical cross-linking of wart and recombinant (baculovirus-expressed) HPV 1 E4 protein indicated that in solution this viral protein forms complexes consistent in size with either trimers or tetramers. These complexes were resistant to urea denaturation and are not dependent on the formation of disulphide linkages. A mutant protein containing a deletion of residues 110 to 115 was unable to form oligomers following cross-linking supporting a role for this region in mediating E4:E4 interactions. We conclude that oligomerisation of the HPV 1 E4 protein is likely to be mediated by carboxy-terminal residues and that conserved hydrophobic residues of this domain play a major role in E4 oligomerisation.
Ashmole
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I
Cancer Research Campaign Institute for Cancer Studies, Medical School, University of Birmingham, United Kingdom.
Gallimore
P H
PH
Roberts
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S
D000595:Amino Acid Sequence; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D027383:Papillomaviridae / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D009856:Oncogene Proteins, Viral; D014764:Viral Proteins; C056651:oncogene protein E4, human papilloma virus type 1